Cover Page. The handle holds various files of this Leiden University dissertation.

Transcription

1 Cover Page The handle holds various files of this Leiden University dissertation. Author: Cunha Carvalho de Miranda, Noel Filipe da Title: Mismatch repair and MUTYH deficient colorectal cancers : at the crossroad of genomic stability and immune escape Issue Date:

2 Summary Nederlandse samenvatting Curriculum Vitae List of publications 8

3 116

4 Summary SUMMARY The works presented in this thesis have focused on the study of specific subtypes of colorectal cancer that arise in a context of deficiency of one of the following DNA repair systems: mismatch repair or base excision repair. Colorectal cancer is one of the most common and deadliest cancers in the world. It is a heterogeneous disease that comprises a range of malignancies that develop according to various genetic/oncogenic pathways. DNA mismatch repair deficient cancers comprise up to 20% of all colorectal cancers and are characterized for exhibiting a microsatellite instability-high (MSI-H) phenotype. The latter refers to the widespread accumulation of small insertions and deletions at microsatellite sequences, throughout the genome. MSI-H colorectal cancers can develop sporadically or due to hereditary mutations in one of the mismatch repair genes (Lynch syndrome). Base excision repair deficient colorectal cancers are caused by hereditary mutations in the MUTYH gene. Carriers of biallelic mutations in MUTYH are at increased risk of developing colorectal polyps and cancer and this syndrome is referred to as MUTYH-associated polyposis (MAP). MAP cancers constitute less than 1% of all diagnosed colorectal cancers. MSI-H colorectal cancers often carry more mutations in their coding genome than the remaining subtypes of colorectal cancer, a consequence of their constitutive DNA repair deficiency. As mutations eventually translate to the protein level, abnormal proteins are generated that can be recognized by the immune system. Effector immune cells, such as cytotoxic CD8+ T cells, have the capacity of eliminating cancer cells that present such proteins and, therefore, tumors must adopt mechanisms that allow them to escape destruction. Among those mechanisms, one of the most important is the loss of expression of HLA class I, the molecule responsible for presenting endogenous peptides to cytotoxic CD8+ T cells. Although the loss of HLA class I expression was previously described as a common event during colorectal carcinogenesis, we and others hypothesized that it was more likely to occur in MSI-H tumors due to their hypermutated genomes. Additionally, MSI-H cancers are notable for presenting a conspicuous immune cell infiltrate in their tissues which reflects a strong anti-tumor immune reaction. Accordingly, in chapter 2, we describe that HLA class I alterations are clearly more frequent in MSI-H tumors than in the remaining spectrum of colorectal malignancies. Loss of expression of HLA class I can occur through a number of mechanisms that include mutations in the HLA class I genes themselves, mutations in the gene encoding for the β chain (β2m), and mutations in molecules that are involved in the processing of the proteins to be loaded on HLA class I, denominated by antigen processing machinery. Also in the work included in chapter 2 we discovered that HLA class I loss was more likely to be associated with deficiencies in members of the antigen processing machinery in sporadic MSI-H cancers than in their hereditary counterpart. Conversely, MSI-H tumors from Lynch syndrome patients were most often affected by deficiencies in the β2m molecule. It is known that deficiencies in β2m or in antigen processing machinery members have different consequences but the reason why sporadic and hereditary MSI-H cancers target one or the other are still elusive. Although it was not yet established whether MAP colorectal cancers carry a higher number of mutations than non-msi-h colorectal tumors, we considered it to be a logical assumption due to their background of base excision repair deficiency. MAP cancers also present evidence for an anti-tumor immune reaction which suggests that, similar to MSI-H cancers, these tumors are also strongly immunogenic. In such case, a strong selective pressure for adaptation mechanisms that allow them to escape immune surveillance would also be in 117

5 place. In the work presented in chapter 3, we describe that HLA class I loss of expression is indeed a frequent event in MAP colorectal cancers. Interestingly, and as observed for Lynch syndrome cancers, loss of HLA class I expression was most often associated with β2m defects, which appears to be a common mechanism in MAP and Lynch colorectal syndromes. With this work we completed the characterization of HLA class I phenotypes in colorectal cancers that arise in a context of DNA repair deficiency. We concluded that such deficiency is likely to be responsible for the generation of a surplus of mutated proteins in tumors, which in turn activates an immune response that favors the outgrowth of tumor clones with immune escape phenotypes. Assuming that the quality and intensity of an anti-tumor immune response influences the HLA class I phenotype of tumors, we investigated whether this relation could be observed in Lynch colorectal cancers, in chapter 4. Indeed we found that cancers carrying HLA class I defects presented higher amounts of tumor-infiltrating lymphocytes, particularly CD4+ and CD8+ T cells. This observation provides a link between the agents of selective pressure (immune cells) and the positively selected tumor traits (loss of HLA class I expression). Furthermore, the magnitude of lymphocytic infiltration not only correlated with HLA class I phenotypes but also with the tumor stage at diagnosis. Infiltration by activated CD8+ T cells (cytotoxic T cells) was higher in tumors diagnosed at early stages than in advanced cancers, supporting the anti-tumorigenic role of these cells. An elusive immune cell type that expressed granzyme B, thus with cytotoxic potential, was almost exclusively present in HLA class I negative tumors that lacked metastases. These characteristics were reminiscent of NK cells but further investigation with a range of discriminative immune cell markers was inconclusive. The identification of these cells could be of great importance of the management of cancers that have lost HLA class I expression. In chapter 5, we present a study on the most frequently mutated gene in MSI-H cancers, TGFBR2. Until now, mutations in TGFBR2 were considered to result in loss of protein function due to the frameshift, truncating nature of the mutations found. We discovered that cells carrying biallelic, frameshift mutations in TGFBR2 were still capable of transducing TGF-β signaling in a TGFβR2-dependent manner. We proposed that the same mechanism that leads to the generation of mutations at the DNA level is probably responsible for the restoration of TGFBR2 s reading frame at the transcriptional level. Thus, frameshift mutations located at microsatellite sequences have the potential to be reversed to a wild type form at the RNA level. Interestingly, we discovered that MSI-H cancers were apparently selected to retain some sensitivity to TGF-β since the occurrence of biallelic mutations that excluded the possibility of reading frame restoration was rare. We suggest that the contribution of this pathway for the tumorigenesis of MSI-H tumors should be reanalyzed in the light of these findings. In chapter 6, we review our findings on MSI-H and MAP tumors in the light of others findings and propose that the oncogenic processes in these cancers are particularly dependent on the interaction between the tumor cells and their microenvironment. In chapter 7, some final considerations are made and suggestions for continuing research. 118